Biotechnological work carried out on plants aims at generating plants with advantageous novel properties, for example to increase agricultural productivity, to increase the quality in foodstuffs or for producing certain chemicals or pharmaceuticals.
Plastids are organelles within plant cells which have their own genome. They play an essential role in photosynthesis and in amino acid and lipid biosynthesis. The plastids' genome consists of a double-stranded, circular DNA with an average size of from 120 to 160 kb and is present—for example in leaf cells—as approximately 1900 to 50,000 copies per cell (Palmer (1985) Ann Rev Genet 19:325-54). A single plastid has a copy number of approximately from 50 to 100. The term plastids comprises chloroplasts, proplastids, etioplasts, chromoplasts, amyloplasts, leukoplasts and elaioplasts (Heifetz P (2000) Biochimie 82:655-666). The various forms can be converted into one another and all arise from the proplastids. This is why all manifested forms of the plastids comprise the same genetic information. Preference is given in the literature as starting material for the transformation of plastids, however, to green cells, which comprise the chloroplasts as the manifested form.
It is of great economic interest for plant biotechnologists to develop efficient methods for the transformation of plastids (McFadden G (2001) Plant Physiol. 125:50-53). The stable transformation of plastids of higher plants is one of the great challenges.
In the transformation of plastids, the technique of undirected (illegitimate) DNA insertion, which is frequently employed in insertion into the nuclear DNA, has the disadvantage that it is highly likely that an essential gene on the gene-dense plastidic genome is affected, which would frequently be lethal for the plant. The directed insertion of foreign DNA is therefore advantageous in plastids.
Various methods for the directed insertion into the plastidic genome have been described. The first to be described was plastid transformation in green algae (Boynton J E et al. (1988) Science 240: 1534-1538; Blowers A D et al. (1989) Plant Cell 1:123-132), followed later by higher plants such as tobacco (Svab Z et al. (1990) Proc Natl Acad Sci USA 87:8526-8530).
EP-A 0 251 654, U.S. Pat. Nos. 5,932,479, 5,451,513, 5,877,402, WO 01/64024, WO 00/20611, WO 01/42441, WO 99/10513, WO 97/32977, WO 00/28014, WO 00/39313 describe methods and DNA constructs for the transformation of plastids of higher plants, where the DNA to be transformed is introduced into the plastome (plastidic genome) via homologous recombination (“double crossover”). In general, homologous regions of 1000 bp or more on either side of the sequence to be inserted are employed. This rapidly gives rise to large vectors whose handling is not very convenient. Moreover, the transformation efficiency drops. The homologous recombination efficiency drops with the increasing length of the foreign DNA to be integrated. A further disadvantage is the fact that a homologous region which can be utilized for the process of DNA integration by means of double crossover must be identified for each plant species. WO 99/10513 claims the identification of an intergenic DNA sequence with supposedly sufficient homology between the genomes of the chloroplasts of many higher plants, which DNA sequence can thus act as a universal target sequence. However, it has not been demonstrated that this vector can be utilized successfully in species other than tobacco; rather, in WO 01/64024, the same inventor adapts the transformation vector to non-tobacco plant species by using homologous DNA sequences isolated from these plant species. Since only few recombination events result in all of the above-described methods, selection of the recombinant plastidic DNA molecules is required.
The plastid DNA of higher plants is present in the form of up to several thousand copies per cell. To ensure stable integration of foreign DNA, all copies of the plastidic DNA must be modified in the same manner. In plastid transformation, this is referred to as having reached the homotransplastomic state. This state is achieved by what is known as a segregation-and-sorting process, by exerting a continuous selection pressure on the plants. Owing to the continual selection pressure, those plastids in which many copies of the plastidic DNA have already been modified are enriched during cell and plastid division. The selection pressure is maintained until the homotransplastomic state is reached (Guda C et al. (2000) Plant Cell Reports 19:257-262). The modification of all of the copies of the plastidic genome in order to obtain homotransplastomic plants which have incorporated the foreign gene stably into their plastidic genome over generations without addition of a selection agent is a great challenge (Bogorad L (2000) TIBTECH 18:257-263). In addition to the continuous selection pressure, achieving the homotransplastomic state is, if appropriate, ensured by repeatedly regenerating tissue which has already been transformed (Svab Z and Maliga P (1993) Proc Natl Acad Sci USA 90:913-917). However, this procedure limits the plant material which is available for plastid transformation. Coupling, if appropriate, the transgene with another gene which is essential for the survival of the plant is therefore proposed.
In most cases, tissue culture techniques and selection processes cannot be applied universally to all plant species and constitute a substantial limitation of plastid transformation, in particular with regard to the applicability of the method to species other than tobacco. (Kota M et al. (1999) Proc Natl Acad Sci USA 96:1840-1845). A recently published transformation of tomato plastids is based on modifications in the regeneration and selection scheme (Ruf S et al. (2001) Nature Biotech 19:870-875), which, however, are expensive and time-consuming. Another approach aims at reducing the number of plastids per cell and the DNA molecules per plastid so that fewer DNA molecules have to be modified (Bogorad L (2000) TIBTECH 18:257-263). All of the selection and segregation processes are very time-consuming.
WO 99/10513 describes a method in which a plastidic ORI (origin of replication) is localized on the plasmid to be transformed in order to increase, in this manner, the number of copies of the vector to be transformed which are available for integration into the plastidic genome (Guda C et al. (2000) Plant Cell Reports 19:257-262).
The necessity of improving the plastid transformation technique is also mentioned in Heifetz and Tuttle (Heifetz P and Tuttle A M (2001) Curr Opin Plant Biol 4:157-161). WO 00/32799 teaches increasing the efficiency of plastid transformation by employing plants with enlarged plastids. This results in a large plastid surface, through which the DNA to be transformed can enter the plastids with greater ease. However, the mechanism of DNA integration relies, again, on conventional homologous recombination, as was the case in the above-described methods.
A variety of other methods for the sequence-specific integration of DNA—in particular into the nuclear DNA—have been described. A method based on self-splicing group II introns has been described. Self-splicing group II introns are capable of inserting in a sequence-specific fashion, for example into intron-free genes. The sequence-specific hydrolysis of the target DNA is catalyzed by an RNA-protein (ribonucleoprotein) complex. Here, the sequence specificity of the endonuclease function is determined in particular by base pairings being formed between the RNA moiety of the ribonucleoprotein complex and the target DNA. The use of group II introns as vectors for foreign DNA has been discussed. By modifying certain sequences of a group II intron, it was possible to modify the target specificity of the latter. Also, it was possible to insert further sequences into group II introns without destroying functions of the latter (Yang J et al. (1996) Nature 381:332-335; Eickbush T H (1999) Curr Biol 9:R11-R14; Matsuura M et al. (1997) Genes Develop 11:2910-2924; Cousineau B et al. (1998) Cell 94: 451-462). The adaptation to certain target sequences and the determination of the associated rules, however, is laborious and has as yet been elucidated in detail only for the Ll.ltrB intron (Mohr G et al. (2000) Genes Develop 14:559-573). Moreover, the retrohoming efficiency was reduced significantly by the modification, and not every single one of the modified introns tested inserted into the desired target DNA. The disadvantage of the technique is that some positions in the nucleotide sequence are fixed, which limits the choice of the target region in the DNA to be transformed (Guo H et al. (1997) EMBO J. 16:6835-6848). Moreover, the efficiency of the retrohoming process with regard to that of the wild-type intron appears to be diminished. The efficiency of intron insertion at different sites on the genes investigated differed with regard to its level. The work aimed at providing an improved method for the directed insertion of DNA into the nuclear DNA of organisms which permit no efficient homologous recombination (Guo et al. (2000) Science 289:452-456). The experiments described have been carried out extrachromosomally both in the prokaryote E. coli and in human cells. The applicability to the chromosomal DNA of higher organisms or the applicability to plastidic DNA was neither described nor demonstrated. It was merely proposed to attempt the optimization of this system in such a way that insertion into chromosomal DNA of higher eukaryotes can take place. This system is supposed to be an alternative method for higher eukaryotes which lack efficient homologous recombination (Guo et al. (2000) Science 289:452-456). This does not apply to plastids of higher plants, where homologous recombination—at least in the case of individual plastidic DNA molecules—can usually be performed without problems.
Plastid transformation was demonstrated not only in tobacco, but also in potato (Sidorov V A et al. (1999) Plant J 19:209-216; WO 00/28014), petunia (WO 00/28014), rice (Khan M S and Maliga P (1999) Nature Biotech 17:910-915; WO 00/07431; U.S. Pat. No. 6,153,813), Arabidopsis (Sikdar S R et al. (1998) Plant Cell Reports 18: 20-24; WO 97/32977) and oilseed rape (WO 00/39313). (Review article: Bogorad L (2000) TIBTECH 18:257-263). Transplastomic tomato plants have also been described recently (Ruf S et al. (2001) Nature Biotech 19:870-875).
The generation of sequence-specific double-strand breaks with the aid of restriction enzymes in eukaryotic genomes, including plants, has been described (Puchta H (1999) Methods Mol Biol 113:447-451).
WO 96/14408 describes the homing restriction endonuclease I-SceI and various possibilities for its use. An application for inserting DNA sequences into plastidic DNA is not described.
Posfai et al. describe a method for the substitution of genes in the prokaryote E. coli (Posfai G et al. (1999) Nucleic Acids Res 27(22):4409-4415). Here, an intramolecular recombination between the endogenous and the mutated gene takes place in the E. coli genome, which combination is induced by cleaving with the restriction enzyme I-SceI. Recombinations in E. coli proceed markedly more efficiently and, presumably, following different mechanisms than is the case in the nucleus of higher eukaryotes (for example described by Kuzminov A (1999) Microbiol Mol Biol Rev. 63(4):751-813).
“Homing” refers to the phenomenon that two or more copies of a DNA sequence exist in one compartment, where at least one of these two sequences is interrupted by a further DNA sequence, and a copy of the interrupting DNA sequence is subsequently also introduced into the noninterrupted DNA sequence. This phenomenon usually takes the form of intron homing. Here, two or more alleles of one gene exist in one compartment, where at least one of these alleles has no intron. A copy of the intron is subsequently also introduced into the intron-free allele.
Introns in plastidic genes of higher plants have been described (Vogel J et al. (1999) Nucl Acids Res 27:3866-3874; Jenkins B D et al. (1997) Plant Cell 9:283-296; Xu M Q et al. (1990) Science 250: 1566-1570). The splicing of a homologous, unmodified intron with the natural exon regions at an ectopic locus in the plastidic genome has likewise been described (Bock R and Maliga P (1995) Nucl Acids Res 23(13):2544-2547). Experiments of introducing, into plastids of higher plants, heterologous introns which are additionally modified in such a way that they comprise additional genetic information and/or splice in a normatural sequence environment have not been carried out as yet.
Experiments carried out by Eddy and Gold into the homing process in E. coli have demonstrated that certain recombination systems are required. The type of the recombination system of the host is a key variable (Eddy S R and Gold L (1992) Proc Natl Acad Sci USA 89:1544-1547). It was therefore impossible to assume that the naturally occurring homing process of one organism can be applied at will to another organism, in particular when this process probably does not occur naturally in the latter organism.
Dürrenberger et al. describe the induction of an intrachromosomal recombination in chloroplasts of the single-celled green alga Chlamydomonas reinhardtii using the I-CreI homing endonuclease (Dürrenberger F et al. (1996) Nucleic Acid Res 24(17):3323-3331).
The recombination takes place between the endogenous 23S gene and a 23S-cDNA which is inserted into the chromosome of an I-CreI deletion strain and which comprises an I-CreI cleavage site. Double-strand breaks are induced by mating the relevant transgenic organism with an organism which naturally expresses I-CreI. At the point in time of the double-strand break, the foreign DNA is already inserted into the chromosomal DNA, and recombination takes place intramolecularly and not between two separate molecules.
It has been shown recently that a mobile intron which naturally occurs in Chlamydomonas reinhardtii and which also encodes a homing endonuclease can be transformed efficiently into an intron-free copy (Odom O W et al. (2001) Mol Cell Biol 21: 3472-3481). In this work, the increase of the transformation rate was dependent on the presence of the homing endonuclease. In the discussion, it is proposed in general terms and without specific suggestions regarding the implementation, to improve plastid transformation by inducing double-strand breaks. To this end, the recognition regions of rare nucleases were initially to be introduced in a first step, and the subsequent integration event was then to take place at the same locus. More detailed suggestions regarding the manner in which the recognition regions are to be introduced, the type of nucleases and recognition regions which can be used, the way in which the first step and the second step can be designed in actual reality, and the like, are not provided. All that has been shown to date is that the introduction of a homologous intron, into plastids of the alga Chlamydomonas, by means of the homing endonuclease naturally associated with the mobility of the intron did work. Moreover, the results were generated in an algal species. The abovementioned experiments by Eddy and Gold with E. coli, where no mobile group I introns are known, as is the case with the plastids of higher plants, demonstrate that an applicability to heterologous systems is not readily feasible. It is therefore by no means obvious for the skilled worker to apply the observations on the alga Chlamydomonas to higher plants. In contrast, there are a number of suggestions which make such an applicability rather doubtful:    1. Homing systems cannot be applied readily from one system to another (Eddy S R and Gold L (1992) Proc Natl Acad Sci USA 89:1544-1547). The applicability to higher plants is all the more dubious since no homing endonucleases have been identified in those plastidic genomes of higher plants which have already been sequenced (http://megasun.bch.umontreal.ca/ogmp/projects/other/cp_list.html). It can therefore be assumed that the introns found in the plastidic genome of higher plants are not mobile, and that no homing mechanism exists naturally in these genomes.    2. Chlamydomonas only has one plastid per cell, while in cells of higher plants up to 100 plastids are present per cell.    3. The efficiency of conventional plastid transformation in Chlamydomonas exceeds that in higher plants by several orders of magnitude, which suggests that these two systems cannot be compared directly with one another. As regards the regeneration of transplastomic algae or transplastomic plants, the fact that division of the algal plastids is synchronized with the cell cycle, while this is not the case for the plastids of the higher plants, might also play an important role (Sato N (2001) Trends Plant Science 6:151-155).    4. The mechanisms of DNA integration into plastids of Chlamydomonas and of higher plants appear to be fundamentally different. Thus, it has been found that inter-specific plastid transformation (where homologous regions are utilized instead of identical sequences) in Chlamydomonas leads to a marked reduction of the transformation efficiency, which was, however, not observed in tobacco. This also applies analogously to the distance of a molecular marker on the homologous DNA from the heterologous sequence on the transformation plasmid: the closer the molecular marker to the edge of the target region for integration by means of double crossover, the less frequently it is transferred when transformed into Chlamydomonas plastids. In tobacco, multiple recombination mechanisms were observed, but here even molecular markers which were close to the edge of the homologous regions were transferred efficiently into the plastidic genome during transformation (Kavanagh TA et al. (1999) Genetics 152: 1111-1122 and references cited therein).    5. In Chlamydomonas, the plastids of the two parents fuse during hybridization, even in the case of inter-specific hybridization. In Chlamydomonas, plastid fusion is a natural process, and the DNA of the plastids too is mixed and undergoes new recombination. This is why mobile introns in the organelles of these organisms make sense. In contrast, in most of the higher plants, the plastids are inherited uniparentally, so that neither mixing of the plastidic DNA results nor recombinations can occur between the maternal and the paternal plastidic DNA. Even in those plant species in which the plastids are inherited biparentally, no plastid fusion was observed. It can therefore be assumed that natural plastid fusion in higher plants can be ruled out (Hagemann R (1992) plastidic genetics in higher plants; in Cell organelles, editor: Herrmann R G, Springer Verlag, Vienna, pp. 65-96) and that mechanisms like intron homing are either not developed or even suppressed.
Increasing the homologous recombination efficiency within the nuclear DNA with the aid of rare endonucleases has been described for various organisms (Puchta H et al. (1993) Nucleic Acids Research. 21(22):5034-40; Puchta H et al. (1996) Proc Natl Acad Sci USA 93:5055-5060; Rong Y S and Golic K G (2000) Science 28:2013-2018; Jasin M (1996) Trends Genet 12: 224-229). In contrast to plastids, insertion by homologous recombination into the nuclear DNA is problematic and usually takes place owing to random illegitimate integration. This demonstrates that techniques which are established for the nuclear genome cannot necessarily be applied to the plastids. In contrast to the situation regarding the nucleus, integration in plastids of higher plants takes place virtually exclusively, and with high efficiency, via homologous recombination (Bock R and Hagemann R (2000) Progress in Botany 61:76-90; Maliga P et al. (1994) Homologous recombination and integration of foreign DNA in plastids of higher plants. In Homologous recombination and gene silencing in plants. Paszkowski J, ed. (Kluwer Academic publishers), pp. 83-93).
The homologous recombination efficiency for the integration of DNA into the plastome has generally not been thought of as a limiting factor and, in contrast, considered as not being critical. Accordingly, current research into the optimization of plastid transformation does not focus on the optimization of homologous recombination but for example on improved selection markers, improved selection and regeneration techniques and the like. Nevertheless, no essential breakthrough has been achieved to date.